Use of PCR in Diagnosis of Human American Tegumentary Leishmaniasis in Rio de Janeiro, Brazil

Abstract

In Brazil, the most common etiological agent of American tegumentary leishmaniasis is Leishmania(Viannia) braziliensis. In general, diagnostic techniques envisage the visualization of the parasite, but that technique has a low sensitivity. The main purpose of the present work was to evaluate the PCR as a routine tool for the diagnosis of leishmaniasis. Biopsy specimens from cutaneous or mucosal lesions were taken from 230 individuals from areas where Leishmania is endemic: 216 patients who had a clinical picture suggestive ofl leishmaniasis and 14 individuals with cutaneous lesions due to other causes. Each specimen was processed for histopathologic examination, culture, touch preparation, and DNA isolation. Oligonucleotides that amplify the conserved region of the minicircle molecules ofLeishmania were used in a hot-start PCR. While at least one conventional technique was positive for Leishmania for 62% (134 of 216) of the patients, PCR coupled to hybridization was positive for 94% (203 of 216) of the patients. The 14 patients whose clinical picture was not suggestive of leishmaniasis had negative results by all techniques. The impact of the PCR was striking in mucosal disease. While the disease in only 17% (4 of 24) of the patients could be diagnosed by conventional techniques, PCR was positive for 71% (17 of 24) of the patients. Hybridization showed that all cases of disease were caused by parasites belonging to the Viannia subgenus. Altogether, the results indicate that PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and is likely to provide valuable epidemiological information about the disease in countries where it is endemic.

Polymerase chain reaction–miniexon: a promising diagnostic method for mucocutaneous leishmaniasis

“…Furthermore, sensitivity reports vary from 0.2% for direct examination to 69% when various parasite identification methods are combined (direct examination, biopsy, culture, and histopathology), due to low parasite numbers in tissue. [1][2][3][4][5][6] Clinical and histopathologic findings are similar to those observed in other infectious diseases, such as histoplasmosis, leprosy, blastomycosis, syphilis, and tuberculosis. Lymphoma and Wegener's disease are included in the differential diagnosis.…”

“…[24][25][26] Ideally, a diagnosis of mucocutaneous leishmaniasis should be based upon the demonstration of Leishmania 23 Serology may have false positive results due to crossed reactions with other diseases, i.e., trypanosomiases, malaria, erythematous lupus, and toxoplasmosis. 5,30 On clinical practice, the diagnosis is made using clinical, histopathologic, and laboratory criteria. We used a composite reference standard that included these criteria, which we used in our previous study with satisfactory results.…”

“…Consequently, kDNA's performance as a PCR target makes it a good diagnostic tool. 2,4,5,31 However, the kDNA target is not effective for species differentiation, and if PCR results are positive, the authors generally use other markers for species identification. 33,34 Attempts have been made to validate a test that could be useful for diagnosis as well as species typification.…”

“…If both types of samples were included in the analysis, sensitivity was 86.7% (95% CI 72.8-100%), exceeding our previous results. 18 Even though PCR results for parasite identification range from 62 to 100% in the literature, 2,4,5,46 most of the studies only include cases in which they inform the proportion positive for PCR, diagnostic criteria are weak, test interpretations are not blinded, and agreement between observers is not reported.…”

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years

“…Using PCR, genetic investigations can be performed with a broad range of DNA sources, such as amber-conserved insects, mummies, slides, blood or semen [8]. A number of reports have described the use of PCR for amplifying DNA from Plasmodium [9,10], Mycobacterium tuberculosis [11], Cryptosporidium parvum [12] and Leishmania [5][6][7][13][14][15][16][17]. The purpose of the present study was to investigate whether extraction and amplification of Leishmania DNA could be achieved from tissue lesion imprints on Giemsa-stained slides that had been stored for up to 36 years.…”